This application is a xc2xa7371 national phase filing of International Application No. PCT/FR96/00802, filed May 29, 1996.
1. Field of the Invention
The present invention relates to a new polypeptide designated xcex94P62, to its variants, to the corresponding nucleic acid sequences and to their therapeutic uses, in particular in anticancer gene therapy.
2. Description of Related Art
Various genes, referred to as oncogenes and suppressor genes, are involved in the control of cell division. Among these, the ras genes and their products, generally designated p21 proteins, perform a key role in the control of cell proliferation in all the eukaryotic organisms in which they have been sought. In particular, it has been shown that certain specific modifications of these proteins cause them to lose their normal control and lead them to become oncogenic. Thus, a large number of human tumours have been associated with the presence of modified ras genes. Similarly, an overexpression of these p21 proteins can lead to a deregulation of cell proliferation. An understanding of the exact role of these p21 proteins in cells, their mode of functioning and their characteristics hence constitutes a most profitable focus of attention for our understanding of carcinogenesis and the therapeutic approach thereto.
In vivo, the precise nature of the events responsible for transduction of the signal initiated by the p21 proteins is not known. However, an increasing. number of results highlight the multiplicity of effectors which interact directly and preferentially with the active form (bound to GTP) of the ras proteins. Among these effectors, the GAP protein has been the first one to have its involvement in the transduction of the signal documented. It is a cytosol protein, present in all eukaryotic organisms, which possesses the faculty of strongly accelerating the hydrolysis of the GTP bound to the normal protein. It possesses two domains providing for different functions. Its carboxy-terminal end carries the catalytic activity which interacts with the p21 proteins and which increases their GTPase activity. At its other end, downstream of the N-terminal portion, there is a juxtaposition of SH2 and SH3 domains which participate in the transduction of the message and interact with other proteins. Among these proteins, there are two, p62 and p190, of 62 kDa and 190 kDa, respectively, in which the tyrosine is strongly phosphorylated. These two proteins form a specific complex with GAP and are immunoprecipitated by antibodies directed against different epitopes of GAP. It is known, in particular, that the SH2 domains of GAP are the regions in which the interactions of p62 with GAP take place. Amino acids 271 to 443 of p62 contain phosphorylated tyrosines and appear to be involved in these interactions. These same phosphorylations appear, moreover, to participate in interactions between p62 and the adapter GRB2. Moreover, along the whole length of the p62 sequence, proline-rich consensus sites are distributed which participate in the binding to the SH3 domains of the tyrosine kinases of the src family, and also of phospholipase Cxcex3.
The p62 (or alternatively Sam68) protein was identified by Wong et al. (Cell 69 (1992) 551). It contains 443 amino acids, the sequence of which has been described in the literature (see SEQ ID No. 2).
In addition to the features mentioned above, the p62 protein displays several features characteristic of hnRNPs (heterogeneous nuclear ribonucleoproteins):
it is rich in glycines
it possesses regions rich in arginines
furthermore, its amino acids 145 to 247 define a region of strong homology with an hnRNP described previously, GRP33. This region contains a consensus binding site for RNAs which is homologous to the one contained in hnRNP K. This consensus site is designated KH domain (KH=hnRNPK Homologue). The conserved residues are essential to the binding to RNAs, and the impact of the non-integrity of this domain in a pathology has been shown for FMR1, which is the product of the gene associated with mental retardation which is observed in fragile X syndrome (Siomi et al., Cell 77 (1994) 33).
The present invention has its basis, in particular, in the demonstration of the importance of the p62 (Sam68) protein in cell proliferation and death. It is the outcome, more especially, of the demonstration that p62 derivatives are capable of interfering in the process of cell transformation, and in particular of inhibiting the signals transduced by the ras and arc proteins. It is the outcome, in addition, of the especially surprising demonstration that these derivatives are also endowed with apoptotic properties, and hence capable of inducing cell death.
A first subject of the invention hence relates to any p62 derivative capable of at least partially inhibiting the interaction between a GAP protein and p62. Preferably, the derivatives according to the invention are capable of at least partially inhibiting the oncogenic power of the ras and/or arc proteins. Still more preferably, the derivatives according to the invention are capable of inducing cell death by apoptosis. The derivatives according to the invention are also characterized by the loss of the capacity to interact with RNA of p62.
The present invention describes, in particular, the demonstration, cloning and characterization of a natural isoform of the p62 protein. This isoform, designated xcex94p62 (or xcex94Sam68), possesses a deletion in the zone of homology to the GRP33 protein, which covers the KH domain. As a result of this deletion, xcex94p62 does not possess the properties of p62 in their entirety. Thus, xcex94p62 possesses a domain of interaction with GAP and intact GRB2, as well as the various proline-rich sequences which are partners of SH3 (FIG. 1). However, xcex94p62 is no longer capable of interacting with nucleic acids as a result of the deletion of the domain of homology to the GRP33 protein. The Applicant also showed that the transfer of xcex94p62 cDNA in various normal or tumoral cell models impedes the cooperation between p62 and Ras and inhibits the signals transduced by normal and oncogenic Ras proteins. Hence, when overexpressed, xcex94p62 interferes with the processes of proliferation and differentiation and leads, in the different cell models, to cell death by apoptosis.
According to a preferred embodiment, the invention relates more especially to any p62 derivative carrying at least one deletion in the zone of homology to the GRP33 protein. More especially, the derivatives according to the invention contain at least one deletion in the region lying between residues 145 and 247 of the p62 protein as shown in the sequence SEQ ID No. 1, and which covers the KH domain. The deletion advantageously involves more than 10 amino acids, and more preferably involves more than 30 amino acids. It can affect one or several sites within this region, provided the resulting derivative displays the properties described above.
It is especially advantageous for the derivative according to the invention to be a polypeptide comprising all or part of the sequence SEQ ID No. 4 or of a variant of the latter. For the purposes of the invention, the term variant denotes any polypeptide whose structure differs from the sequence SEQ ID No. 4 by one or more modifications of a genetic, biochemical and/or chemical nature. Such modifications can entail, in particular, any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives may be generated for different purposes, such as, in particular, that of increasing the affinity of the peptide for its interaction site, that of improving its levels of production, that of increasing its resistance to proteases or of improving its passage through cell membranes, that of increasing its therapeutic efficacy or of reducing its side effects or that of endowing it with new pharmacokinetic and/or biological properties. Advantageously, the variants comprise deletions or mutations involving amino acids whose presence is not decisive for the activity of the derivative. Such amino acids may be identified, for example, by tests of cellular activity as described in the examples.
As a special preference, the derivatives of the invention retain at least a portion of the p62 protein permitting the interaction with the SH2 domain of GAP. This portion of p62 consists, more especially, of phosphorylated tyrosines localized between residues 200 and 443 of the p62 protein (see SEQ ID No. 2). A preferred derivative according to the invention hence comprises at least (i) a deletion in the region lying between residues 145 and 247 of p62, and (ii) a portion of p62 permitting the interaction with the SH2 domain of GAP. More preferably, the deletion involves residues 1 to 202.
In this connection, the Applicant also showed that derivatives according to the invention displaying especially advantageous properties can consist of polypeptides essentially comprising the region carrying the phosphorylated tyrosines of p62.
An especially preferred example of polypeptide according to the invention is represented by the polypeptide xcex94p62 of sequence SEQ ID No. 4, possessing a deletion of residues 170-208 of the sequence of p62. Another example is represented by the polypeptide p62-C comprising residues 203 to 443 of p62 (sequence SEQ ID No. 6).
The results presented in the present application show, in particular, that xcex94p62 can compete with p62 for GAP. Since GAP is one of the effectors of the Ras proteins, xcex94p62 blocks the mitogenic pathways dependent thereon. When overexpressed by gene transfer (transfection, infection, microinjection, and the like), xcex94p62 induces cell death by apoptosis in normal cells (NIH3T3 and Swiss 3T3 fibroblasts) or tumour cells (H460;HCT116), and is capable of inhibiting the formation of foci induced by ras. This same effect is obtained with the derivative p62-C (essentially comprising the C-terminal portion of xcex94p62, which covers the region lying between amino acids 203 and 443 and which corresponds to the domain of interaction with the SH2 domains of GAP and of GRB2). This C-terminal portion also contains three of the sites of interaction with the SH3 domains, those having most affinity for Fyn. The substantial therapeutic activity of the derivatives according to the invention is associated with their multifarious properties, and in particular their power of titration of the SH3 domains of proteins of the src family (for example fyn), their capacity for inhibition of the recruitment of GRB2 by titrating its SH2 domain and their capacity for inhibition of the effector function of the GAP protein for the Ras dependent pathways of signalling.
Another subject of the present invention relates to any nucleic acid coding for a polypeptide as defined above.
The nucleic acid according to the invention can be a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In addition, it can be a genomic DNA (gDNA) or complementary DNA (cDNA). It may be of human, animal, viral, synthetic or semi-synthetic origin. It may be obtained in various ways, and in particular by chemical synthesis using the sequences presented in the application and, for example, a nucleic acid synthesizer. It may also be obtained by the screening of libraries by means of specific probes, in particular such as the ones described in the application (see sequences SEQ ID No. 9 and 10, for example). It may also be obtained by mixed techniques including chemical modification (elongation, deletion, substitution, and the like) of sequences screened from libraries. Generally speaking, the nucleic acids of the invention may be prepared according to any technique known to a person skilled in the art.
Preferably, the nucleic acid according to the invention is a cDNA or an RNA.
The nucleic acid according to the invention is advantageously chosen from:
(a) all or part of the sequence SEQ ID No. 3 or SEQ ID No. 5 or of their complementary strand,
(b) any sequence hybridizing with the sequences (a) and coding for a derivative according to the invention,
(c) the variants of (a) and (b) resulting from the degeneracy of the genetic code.
As mentioned above, the Applicant has now isolated and characterized new nucleic acid sequences coding for polypeptides derived from p62, having altogether exceptional antiproliferative and apoptotic properties. These nucleic acids may now be used as therapeutic agents for producing in cells derivatives according to the invention capable of destroying or correcting cellular dysfunctions. To this end, the present invention also relates to any expression cassette comprising a nucleic acid as defined above, a promoter permitting its expression and a transcription termination signal. The promoter is advantageously chosen from promoters which are functional in mammalian, preferably human, cells. More preferably, it is a promoter permitting the expression of a nucleic acid in a hyperproliferative cell (cancer cell, restenosis, and the like). In this connection, various promoters may be used. For example, the p62 gene""s own promoter may be used. Promoter regions of different origin (responsible for the expression of other proteins, or even synthetic regions) may also be used. Thus, it is possible to use any promoter or derived sequence that stimulates or represses the transcription of a gene, specifically or otherwise, inducibly or otherwise, strongly or weakly. The promoter sequences of eukaryotic or viral genes may be mentioned in particular. For example, the promoter sequences may be ones originating from the genome of the target cell. Among the eukaryotic promoters, it is possible to use, in particular, ubiquitous promoters (promoter of the HPRT, PGK, xcex1-actin, tubulin, and the like, genes), promoters of intermediate filaments (promoter of the GFAP, desmin, vimentin, neurofilaments, keratin, and the like, genes), promoters of therapeutic genes (for example the promoter of the MDR, CFTR, factor VIII, ApoAI, and the like, genes), tissue-specific promoters (promoter of the pyruvate kinase, villin, intestinal fatty acid binding protein, smooth muscle xcex1-actin, or the like, gene) or alternatively promoters that respond to a stimulus (steroid hormone receptor, retinoic acid receptor, and the like). Similarly, promoter sequences originating from the genome of a virus may be used, such as, for example, the promoters of the adenovirus E1A and MLP genes, the CMV early promoter or alternatively the RSV LTR promoter, and the like. In addition, these promoter regions may be modified by adding activating or regulatory sequences, or sequences permitting a tissue-specific or -preponderant expression.
The present invention now provides new therapeutic agents which make it possible, as a result of their antiproliferative and/or apoptotic properties, to interfere with a large number of cellular dysfunctions. For this purpose, the nucleic acids or cassettes according to the invention may be injected as they are at the site to be treated, or incubated directly with the cells to be destroyed or treated. It has, in effect, been reported that naked nucleic acids can enter cells without a special vector. Nevertheless, it is preferable in the context of the present invention to use an administration vector, enabling (i) the efficacy of cell penetration, (ii) targeting and (iii) extra- and intracellular stability to be improved.
According to an especially preferred embodiment of the present invention, the nucleic acid or cassette is incorporated in a vector. The vector used may be of chemical origin (liposome, nanoparticle, peptide complex, cationic polymers or lipids, and the like) viral origin (retrovirus, adenovirus, herpesvirus, AAV, vaccinia virus, and the like) or plasmid origin.
The use of viral vectors is based on the natural transfection properties of viruses. It is thus possible to use, for example, adenoviruses, herpesviruses, retroviruses and adeno-associated viruses. These vectors prove especially efficacious is from the standpoint of transfection. In this connection, a preferred subject according to the invention lies in a defective recombinant retrovirus whose genome comprises a nucleic acid as defined above. Another particular subject of the invention lies in a defective recombinant adenovirus whose genome comprises a nucleic acid as defined above.
The vector according to the invention can also be a non-viral agent capable of promoting the transfer of nucleic acids into eukaryotic cells and their expression therein. Synthetic or natural chemical or biochemical vectors represent an advantageous alternative to natural viruses, especially on grounds of convenience and safety and also on account of the absence of theoretical limit regarding the size of the DNA to be transfected. These synthetic vectors have two main functions, to compact the nucleic acid to be transfected and to promote its binding to the cell as well as its passage through the plasma membrane and, where appropriate, the two nuclear membranes. To mitigate the polyanionic nature of nucleic acids, the non-viral vectors all possess polycationic charges.
The nucleic acid or vector used in the present invention may be formulated for the purpose of administration topically, orally, parenterally, intranasally, intravenously, intramuscularly, subcutaneously, intraocularly, transdermally and the like. Preferably, the nucleic acid or vector is used in an injectable form. It may hence be mixed with any pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection at the site to be treated. The formulation may comprise, in particular, isotonic sterile solutions, or dry, in particular lyophilized, compositions which, on addition of sterilized water or of physiological saline as appropriate, enable injectable solutions to be made up. A direct injection of the nucleic acid into the patient""s tumour is advantageous, since it enables the therapeutic effect to be concentrated in the tissues affected. The doses of nucleic acid used may be adapted in accordance with various parameters, and in particular in accordance with the gene, the vector, the mode of administration used, the pathology in question or alternatively the desired length of treatment.
The invention also relates to any pharmaceutical composition comprising at least one nucleic acid.
It also relates to any pharmaceutical composition comprising at least one vector as defined above.
It also relates to any pharmaceutical composition comprising at least one p62 derivative as defined above.
As a result of their antiproliferative properties, the pharmaceutical compositions according to the invention are most especially well suited to the treatment of hyperproliferative disorders such as, in particular, cancers and restenosis. Thus the present invention provides an especially effective method for the destruction of cells, in particular hyperproliferative cells. It may be used in vitro or ex vivo. Ex vivo, it consists essentially in incubating the cells in the presence of one or more nucleic acids (or of a vector or cassette or of the derivative directly). In vivo, it consists in administering to the body an active amount of a vector (or cassette) according to the invention, preferably directly at the site to be treated (tumour in particular). In this connection, the subject of the invention is also a method of destruction of hyperproliferative cells, comprising the bringing of the said cells or of a portion of them into contact with a nucleic acid as defined above.
The present invention is advantageously used in vivo for the destruction of hyperproliferating (i.e. abnormally proliferating) cells. It is thus applicable to the destruction of tumour cells or smooth muscle cells of the vascular wall (restenosis). It is most especially suitable for the treatment of cancers in which an activated oncbgene is involved. As an example, there may be mentioned adenocarcinoma of the colon, thyroid cancer, carcinoma of the lung, myeloid leukaemia, colorectal cancer, breast cancer, lung cancer, stomach cancer, cancer of the oesophagus, B lymphoma, ovarian cancer, bladder cancer, glioblastoma, hepatocarcinoma, cancer of the bone, skin and pancreas or alternatively kidney and prostate cancer, and the like.
The products of the invention are also useful for the identification of other partners of the pathways of signalling of oncogenes, by testing for inhibitors, agonists, competitors or molecules that interact in vivo with these products.
Moreover, the invention also relates to antisense sequences whose expression a target cell enables the transcription and/or translation of cellular mRNAs coding for p62 or xcex94p62 to be controlled. Such sequences can, for example, be transcribed in the target cell into RNAs complementary to the xcex94p62 or p62 cellular mRNAs, and can thus block their translation into protein, according to the technique described in Patent EP 140,308. Such sequences can consist of all or part of the nucleic acid sequences SEQ ID NO. 1, 3 or 5, transcribed in the reverse orientation.
The present invention also relates to the use of any compound capable of inducing the expression or overexpression of xcex94p62 in a cell, for the preparation of a pharmaceutical composition intended for the treatment of hyperproliferative disorders.
The present invention will be described in greater detail by means of the examples which follow, which are to be considered to be illustrative and non-limiting.